The growing threat of antibiotic resistant microorganisms has prompted a search for new drug targets as well as for a better understanding of mechanisms of resistance in order to design effective inhibitors to combat antibiotic resistance and reduce the further spread of resistant bacteria. This application centers on the recently discovered kinases FomA and FomB that represent a novel mechanism of resistance to the widely used antibiotic fosfomycin. These two novel proteins inactivate fosfomycin through phosphorylation. In contrast to all previously known fosfomycin resistance proteins (FosA, FosB and FosX type) the FomA and FomB proteins do not belong to the vicinal chelate superfamily of enzymes, but are members of the superfamily of kinases. They show low sequence identity (~25% at the most) to any known proteins and no sequence identity to the previously described fosfomycin resistance proteins of FosA, FosB or FosX type. In addition, the phosphorylation and diphosphorylation of the phosphonate function has proven to be reversible. The structural studies will characterize the molecular fold of these two unusual kinases and provide a basis for understanding the mechanism of resistance by phosphorylation. Such knowledge will guide the design of inhibitors that will be effective against antibiotic resistance. PUBLIC HEALTH RELEVANCE The basis of the proposal is crystal structure determination of two novel kinases FomA and FomB that confer high-level resistance to common antibiotic fosfomycin. The goal of the proposal is to understand the mechanism of fosfomycin inactivation in order to design inhibitors that will be effective against antibiotic resistance.